Chromatography is a technique used to separate mixtures into individual components. It works by exploiting differences in how fast components migrate and adhere to stationary and mobile phases. There are several types including paper chromatography, thin layer chromatography, gas chromatography and liquid chromatography. Paper chromatography involves using a stationary phase like filter paper and a mobile phase like water or solvent to separate ink components by capillary action. Thin layer chromatography uses a thin silica plate as the stationary phase and a solvent as the mobile phase to separate components into spots visualized by color reagents. Chromatography is used in various applications such as forensic analysis and identifying biological molecules.

2. Chromatography
Presented By: Raheel, Shabir, Hafiz Shahid
Presented To: Sir Zahoor Ahmad

3. What is Chromatography
• Chromatography is a technique for separating
mixtures into their components in order to analyze,
identify, purify, and/or quantify the mixture or
components.
Separate
ComponentsMixture
• Analyze
• Identify
• Purify
• Quantify

4. Detailed Definition:
Chromatography is a laboratory technique that separates
components within a mixture by using the differential affinities of the
components for a mobile medium and for a stationary adsorbing medium
through which they pass.
Definition of Chromatography
Mixture Components
Separation
Stationary Phase

5. • Capillary Action – the movement of liquid within the
spaces of a porous material due to the forces of adhesion,
cohesion, and surface tension. The liquid is able to move up
the filter paper because its attraction to itself is stronger than
the force of gravity.
• Solubility – the degree to which a material (solute)
dissolves into a solvent. Solutes dissolve into solvents that
have similar properties. (Like dissolves like) This allows
different solutes to be separated by different combinations of
solvents.
Separation of components depends on both their solubility in
the mobile phase and their differential affinity to the mobile
phase and the stationary phase.

6. • Liquid Chromatography
• Gas Chromatography
• Paper Chromatography
• Thin-Layer Chromatography
Types of Chromatography

9. Three common ways for carrying
out the Paper Chromatography
Ascending
Descending
Radial/Circular

10. Ascending Paper Chromatography

11. Descending Paper Chromatography

12. Radial/Circular Paper Chromatography
1.
Six sources of water-soluble
black ink were used in the
chromatographic separation.
2.
The six inks were dotted onto the
filter paper and labeled with a
pencil.
3.
After the wick is inserted into
the center of the paper, the
water enters the filter paper
from a Petri dish. The water
goes up the wick and spreads
out radially due to capillary
action.

13. Rf Values Calculation
RF value (in chromatography) The distance travelled by a given component divided
by the distance travelled by the solvent front. For example, the photosynthetic
pigments of an organism and the metabolites of a drug excreted in the urine can be
identified by their RFvalues in paper or thin-layer chromatography.

14. Uses of paper Chromatography
• Identification of unknown organic and inorganic
compounds from a mixture.
• In forensic studies paper chromatography is used in
crime scene investigation and DNA and RNA
sequencing along with other studies.
• Paper chromatography is used as an analytical
chemistry technique for identifying and separating
colored mixtures like pigments.
• Sugars, amino acids, lipids and nucleic acids and other
bimolecular can be easily identified by spraying with
appropriate reagents to detect these specific
compounds.

15. Thin Layer Chromatography

16. Thin Layer Chromatography
One of analysis method that is used to identify the
unknown compounds and to determine the purity of
mixture.
• This method is simple, rapid and cheap
• Widely used in pharmaceutical & food stuff
industry.
• -A plate of TLC can be made from aluminium or
glass which is coated by a solid matter as a
stationary phase.
- The coated material has 0.1-0.3mm in thickness

17. STATIONARY PHASE
• Silica is commonly used as stationary
phase
• The separation of sample mixture will
be depent on the polarity of sample.

18. – As the mobile phase rises up the TLC plate by
capillary action, the components dissolve in
the solvent and move up the TLC plate.
– Individual components move up at different
rates, depending on intermolecular forces
between the component and the silica gel
stationary phase and the component and the
mobile phase.
The stationary phase is SiO2 and is very “polar”.
It is capable of strong dipole-dipole and H-bond donating and accepting
interactions with the “analytes” (the components being analyzed).
More polar analytes interact more strongly with the stationary phase in move
very slowly up the TLC plate.
By comparison, the mobile phase is relatively nonpolar and is capable
of interacting with analytes by stronger London forces, as well as by dipole-
dipole and H-bonding.
More nonpolar analytes interact less strongly with the polar silica gel and more
strongly with the less polar mobile phase and move higher up the TLC plate.

19. MOBILE PHASE
• The ability of mobile phase to move up
is depent on the polarity itself
• Volatile organic solvents is preferably
used as as mobile phase.

20. METHOD

21. • 1.Developing Container
Preparation.
• 2. TLC Plate Preparation.
• 3.Spotting’ TLC plates.
• 4.‘Develop the plate’.
• 5. Detection of spots

22. 6. DETECTION OF SPOT
1) Iodination-put the plate in which the spots face to
the iodine crystall and see what is the spot color
changing
2) Ninhydrin:
-specific identification of amino acid compounds.
- Ninhydrin solution will show a purple spot when
it is sprayed to the amino acid spot.
3) KMnO4
used to identify a reducing agent such as glucose,
fructose, vitamin C and others.
4) Alkaline tetrazolium blue
specificaly used for corticosteroid identification

23. Thin-Layer Chromatography:
Determination of Rf Values
Rf of component A =
dA / dS
Rf of component B =
dB / dS
solvent front
component B
component A
origin
dS
dB
dA
The Rf value is a decimal
fraction, generally only
reported to two decimal
places

24. TLC Compared to Paper
Chromatography
Precise and effective
More stable toward various organic solvents

25. Advantages
• Cheap
• Simple
• The developing can be monitored visually
• Able to use various chemical as a detector
•

26. Refrences
1. R.A.Day, Jr. A.L.Undewood(1987). Analisis
Kualititatif. Edisi ke Empat, ms: 474-529.
2. David G. Watson(2005). Pharmaceutical
analysis. Edisi ke-2, ms 315-331
3. http//orgchem.colorado.edu/hndbooksuppo
rt/TLC/TLCprocedure.html